The cap-binding activity of eukaryotic initiation factor (eIF) 4F is down-regulated during mitosis, heat shock and viral infection, shutting off cap-dependent translation but permitting selective cap- independent translation of a few mRNAs. Initiation on a subset of such mRNAs results from internal ribosomal entry. The objectives of this proposal is to characterize this initiation mechanism using encephalomyocarditis virus (EMCV) RNA a model EMCV initiation was reconstituted in vitro from tRNA, mRNA, ribosomal subunits and purified native and recombinant factors: internal initiation required only canonical eIFs 2, 3 and the 4A and 4G subunits of 4F. It exploits a novel specific RNA-binding activity of 4G rather than the cap-binding activity of the 4E subunit. This fully reconstituted reaction will not be used to dissect individual steps in the initiation process, thereby providing insights into its mechanism and mode of regulation. (1) It will be used to determine if canonical eIFs are sufficient to mediate internal initiation on other iRESs and whether they specifically bind 4G. (2) Sequential binding of eIFs to the EMCV IRES will be analyzed by chemical/enzymatic footprinting to identify binding sites, cofactor requirements for binding and conformational changes in the IRES caused by or required for binding. (3) Determinants and functions of 4G's RNA-binding activity in cap-dependent and independent initiation will be investigated. Regulation of 4F's cap and RNA-binding activities could provide a switch between ap-dependent and independent modes of initiation. (4) Biochemical and genetic techniques will be used to identify interactions between eIFs that (a) prepare mRNA conformation so as to permit and (b) that subsequently promote and stabilize ribosomal binding to the IRES. The ability to reconstitute translation initiation on synthetic mRNA using recombinant factors will enable the functional importance of many of these interactions to be determined.